1. Field of the Invention
The present invention relates to a method for processing of reaction solutions, containing whole-cell catalysts, an aqueous component and an organic component, wherein the organic component contains the product to be enriched.
2. Description of the Related Art
Biotransformations with whole-cell catalysts (among other things, also called whole-cell biocatalysts) have proved to be highly attractive production techniques for the synthesis of fine chemicals (for a survey, see e.g.: S. Buchholz, H. Gröger, “Ganzzellbiokatalyse” in: Angewandte Mikrobiologie, Chapter 8, Springer-Verlag, Berlin, 2006, p. 161f). In comparison with isolated, especially purified enzymes, recombinant whole-cell catalysts often represent a more cost-effective type of catalyst, since processing and purification steps are omitted in the production of the whole-cell catalyst by the direct use of the biomass obtained in fermentation (in contrast to the use of isolated enzymes). Furthermore, the use of whole-cell catalysts in redox reactions permits the reactions to be carried out without addition of external amounts of expensive cofactor (see WO/2005/121350), whereas addition of a cofactor is necessary in reactions with isolated enzymes.
At present various methods are known for the processing of biotransformation reaction solutions containing whole-cell catalysts. A general problem is the formation of emulsions in the extraction operation, together with very long, economically unacceptable processing times, especially extraction and/or filtration times, of for example several hours even at the laboratory scale or even impossibility of separating such mixtures, especially as a result of formation of stable emulsions. The work of G. Jörg, K. Leppchen, T. Daussmann, M. Bertau, Chem. Ing. Techn. 2004, 76, 1739-1742 may be cited as a typical example of this, according to which the chief complication in extractive processing of the whole-cell biotransformation with high cell density is the formation of stable gels and slimes in contact with the organic solvent. The corresponding consequence of this is the need for subsequent purification of the product by distillation, and large losses of yield in downstream processing, accompanied by reduced overall economy of the process and high production costs.
Furthermore, in a survey from the year 2004 on downstream processing of biotransformation solutions, Yazbeck et al. (D. R. Yazbeck, C. A. Martinez, S. Hu, J. Tao, Tetrahedron: Asymmetry 2004, 15, 2757-2763) refer in detail to the often complicated product isolation as a result of the formation of emulsions. It is pointed out that not many user-friendly technologies are available for avoiding this problem, and there is an increasing demand for better ways of improving downstream processing in such systems.
Accordingly, generally there is considerable interest in processing methods that lead to avoidance of the aforementioned limitations. To date, the following methods have been developed:
In the whole-cell-catalyzed synthesis of 2-phenylethanol, in order to avoid the formation of emulsions, which even occurs at low substrate—or product concentrations of <10 g/L, Serp et al. use synthetic resin immobilizates, in which an organic solvent (dibutyl sebacate) is enclosed, for the extraction (D. Serp, U. von Stockar, I. W. Marison, Biotechnol. Bioeng. 2003, 82, 103-110). The product 2-phenylethanol is removed in situ from the reaction medium by being absorbed in the synthetic resin immobilizates. However, this method using organic solvents enclosed in immobilizates is very expensive and cost-intensive. Moreover, the product concentrations used in the solutions are low (<10 g/L).
Another method for processing emulsions consisting of an organic phase, aqueous phase and whole-cell catalysts uses several hydrocyclones arranged in series (L.-Q. Yu, T. A. Meyer, B. R. Folsom, E P 900 113, 1999). The overflow from one hydrocyclone is always transferred to the next hydrocyclone, so that the aqueous phase can be separated from the organic phase and the biocatalyst. However, this method requires high capital expenditure and is correspondingly expensive.
Recently, Bertau et al. showed that it is possible to avoid stable gels and slimes in contact with the organic solvent by using suitable enzymes as demulsifiers (G. Jörg, K. Leppchen, T. Daussmann, M. Bertau, Chem. Ing. Techn. 2004, 76, 1739-1742 and G. Jörg, K. Leppchen, T. Daussmann, M. Bertau, Biotechnol. Bioeng. 2004, 87, 525-536). In this case the problem of gel formation could be suppressed by bioemulsifiers derived from the whole-cell catalyst, which are released by the microorganisms into the medium. A disadvantage, however, is the need for a further, additional enzymatic component, especially if this is only available at great expense. Another problem is the occurrence of side reactions, caused by the proteases that are added (G. Jörg, K. Leppchen, T. Daussmann, M. Bertau, Chem. Ing. Techn. 2004, 76, 1739-1742). There is preferably cleavage of e.g. ester groups, but also of amide groups.
As an alternative, the use of a filter aid after biotransformation has been carried out is reported by Hanson et al. (R. L. Hanson, S. Goldberg, A. Goswami, T. P. Tully, R. N. Patel, Adv. Synth. Catal. 2005, 347, 1073-1080). According to this method, reduction of an organic ketone substrate is carried out with a whole-cell catalyst at a neutral pH (pH 7) and an amount of substrate of about 20 g/L of reaction volume using the high-priced filter aid AMBERLITE XAD-16 in a 10-fold amount relative to the amount of organic substrate used. Although the use of AMBERLITE XAD-16 proved suitable, direct extraction with ethyl-ethyl acetate or MTBE in the absence of such a filter aid did not prove practicable and led to emulsion problems. Drawbacks of the method of Hanson et al. are, however, limitations such as the high consumption of high-priced AMBERLITE XAD-16 as filter aid at a factor of 10 relative to the organic component, even at a low proportion of organic ketone substrate of only about 20 g/L.